Cutting edge: A double-mutant knockin of the CD28 YMNM and PYAP motifs reveals a critical role for the YMNM motif in regulation of T cell proliferation and Bcl-xL expression.

CD28 is a critical regulator of T cell function, augmenting proliferation, cytokine secretion, and cell survival. Our previous work using knockin mice expressing point mutations in CD28 demonstrated that the distal proline motif was primarily responsible for much of CD28 function, whereas in marked contrast to prior studies, mutation of the PI3K-binding motif had little discernible effect. In this study, we examined the phenotype of mice in which both motifs are simultaneously mutated. We found that mutation of the PYAP motif unmasks a critical role for the proximal tyrosine motif in regulating T cell proliferation and expression of Bcl-xL but not cytokine secretion. In addition, we demonstrated that, although function is more severely impaired in the double mutant than in either single mutant, there remained residual CD28-dependent responses, definitively establishing that additional motifs can partially mediate CD28 function.

A detailed phenotypic analysis of immune cell populations in the bronchoalveolar lavage fluid of atopic asthmatics after segmental allergen challenge.

BACKGROUND: Atopic asthma is characterized by intermittent exacerbations triggered by exposure to allergen. Exacerbations are characterized by an acute inflammatory reaction in the airways, with recruitment of both innate and adaptive immune cells. These cell populations as well as soluble factors are critical for initiating and controlling the inflammatory processes in allergic asthma. Detailed data on the numbers and types of cells recruited following allergen challenge is lacking. In this paper we present an extensive phenotypic analysis of the inflammatory cell infiltrate present in the bronchoalveolar lavage (BAL) fluid following bronchoscopically directed allergen challenge in mild atopic asthmatics. METHODS: A re-analysis of pooled data obtained prior to intervention in our randomized, placebo controlled, double blinded study (costimulation inhibition in asthma trial [CIA]) was performed. Twenty-four subjects underwent bronchoscopically directed segmental allergen challenge followed by BAL collection 48 hours later. The BAL fluid was analyzed by multi-color flow cytometry for immune cell populations and multi-plex ELISA for cytokine detection. RESULTS: Allergen instillation induced pro-inflammatory cytokines (IL-6) and immune modulating cytokines (IL-2, IFN-?, and IL-10) along with an increase in lymphocytes and suppressor cells (Tregs and MDSC). Interestingly, membrane expression of CD30 was identified on lymphocytes, especially Tregs, but not eosinophils. Soluble CD30 was also detected in the BAL fluid after allergen challenge in adult atopic asthmatics. CONCLUSIONS: After segmental allergen challenge of adult atopic asthmatics, cell types associated with a pro-inflammatory as well as an anti-inflammatory response are detected within the BAL fluid of the lung.

CTLA4Ig inhibits effector T cells through regulatory T cells and TGF-ß.

The CD28 costimulatory receptor is a critical regulator of T cell function, making it an attractive therapeutic target for the treatment of immune-mediated diseases. CTLA4Ig, now approved for use in humans, prevents naive T cell activation by binding to B7 proteins and blocking engagement of CD28. However, CTLA4Ig suppresses inflammation even if administered when disease is established, suggesting alternative mechanisms. We identified a novel, CD28-independent mechanism by which CTLA4Ig inhibits activated T cells. We show that in vitro, CTLA4Ig synergizes with NO from bone marrow-derived macrophages to inhibit T cell proliferation. Depletion of regulatory T cells (Tregs) or interference with TGF-ß signaling abrogated the inhibitory effect of CTLA4Ig. Parallel in vivo experiments using an allergic airway inflammation model demonstrated that this novel mechanism required both macrophages and regulatory T cells. Furthermore, CTLA4Ig was ineffective in SMAD3-deficient mice, supporting a requirement for TGF-ß signaling. Thus, in addition to preventing naive T cells from being fully activated, CTLA4Ig can turn off already activated effector T cells by an NO/regulatory T cell/TGF-ß-dependent pathway. This mechanism is similar to cell-extrinsic effects of endogenous CTLA4 and may be particularly important in the ability of CTLA4Ig to treat chronic inflammatory disease.

Experimental advances in understanding allergic airway inflammation.

Asthma is largely an inflammatory disease, with the development of T cell mediated inflammation in the lung following exposure to allergen or other precipitating factors. Currently, the major therapies for this disease are directed either at relief of bronchoconstriction (ie beta-agonists) or are non-specific immunomodulators (ie, corticosteroids). While much attention has been paid to factors that regulate the initiation of an inflammatory response, chronic inflammation may also be due to defects in regulatory mechanisms that limit or terminate immune responses. In this review, we explore the elements controlling both the recruitment of T cells to the lung and their function. Possibilities for future therapeutic intervention are highlighted.

A randomized controlled trial to evaluate inhibition of T-cell costimulation in allergen-induced airway inflammation.

RATIONALE: T lymphocytes are important in the pathogenesis of allergic asthma. Costimulation through CD28 is critical for optimal activation of T cells, and inhibition of this pathway with CTLA4Ig has been shown to be effective in preventing airway inflammation and hyperresponsiveness in animal models of asthma. Abatacept, a humanized version of CTLA4Ig, has been approved for treatment of rheumatoid arthritis, providing the opportunity to test whether inhibition of costimulation is an effective strategy to treat people with asthma. OBJECTIVES: To determine if 3 months of treatment with abatacept reduced allergen-induced airway inflammation in people with mild atopic asthma. METHODS: Randomized, placebo-controlled, double-blinded study. Bronchoscopically directed segmental allergen challenge was performed on 24 subjects followed by bronchoalveolar lavage 48 hours later. Subjects were randomized 1:1 to receive abatacept or placebo, followed by a second allergen challenge protocol after 3 months of study drug. MEASUREMENTS AND MAIN RESULTS: There was no significant reduction in allergen-induced eosinophilic inflammation in the abatacept-treated group compared with placebo (17.71% ± 17.25% vs. 46.39% ± 29.21%; P = 0.26). In addition, we did not detect an effect of abatacept on FEV1, provocative concentration of methacholine sufficient to induce a 20% decline in FEV1, or asthma symptoms. Subjects treated with abatacept had an increased percentage of naive and a corresponding decrease in memory CD4(+) T cells in the blood compared with placebo. CONCLUSIONS: Inhibition of CD28-mediated costimulation with abatacept does not seem to alter the inflammatory response to segmental allergen challenge or clinical measures of asthma symptoms in people with mild atopic asthma. Clinical trial registered with ClinicalTrials.gov (NCT 00784459).

Airway epithelial cells suppress T cell proliferation by an IFNγ/STAT1/TGFß-dependent mechanism.

Organ-specific regulation of immune responses relies on the exchange of information between nonimmune and immune cells. In a primary culture model of the lung airway, we demonstrate that T cell proliferation is potently inhibited by airway epithelial cells (ECs). This is mediated by activation of the IFNγ/STAT1 pathway in the EC and transforming growth factor-ß (TGFß)-dependent suppression of T cell proliferation. In this way, the EC can restrict the expansion of T cells. Given the constant exposure of the airway to inhaled antigen, this may be important in setting a threshold for the initiation of T cell-dependent immune responses and preventing unwanted, chronic inflammation.

CTLA4-Ig inhibits allergic airway inflammation by a novel CD28-independent, nitric oxide synthase-dependent mechanism.

The T-cell response to antigen depends upon coordinate signaling between costimulatory and inhibitory receptors. Altered function of either may underlie the pathophysiology of autoimmune and/or chronic inflammatory diseases and manipulation of these pathways is an important emerging area of therapeutics. We report here that the immunosuppressant drug CTLA4-Ig inhibits the effector phase of allergic airway inflammation through a CD28-independent, nitric oxide synthase dependent mechanism. Using mice deficient in both B and T lymphocyte attenuator (BTLA) and CD28, we demonstrate that simultaneous deficiency of an inhibitory receptor can rescue the in vivo but not the in vitro CD28-deficient phenotype. Furthermore, we demonstrate that inflammation in the CD28/BTLA-double-deficient mice is suppressed by CTLA4-Ig. This suppression is reversed by treatment with the Nitric Oxide Synthase (NOS) inhibitor, N(6)-methyl-L-arginine acetate (L-NMMA). In addition CTLA4-Ig was ineffective at inhibiting inflammation in NOS2-deficient mice when given at the effector phase. Thus, CD28 and BTLA coordinately regulate the in vivo response to inhaled allergen, and CTLA4-Ig binding to B7-proteins inhibits the effector phase of inflammation by a CD28-independent, NOS-dependent mechanism.

CD28 facilitates the generation of Foxp3(-) cytokine responsive regulatory T cell precursors.

The T cell costimulatory molecule CD28 plays an important role in the thymic generation of Foxp3(+) regulatory T cells (Tregs) essential for the maintenance of self-tolerance. In this study, we show that a cell-intrinsic signal from CD28 is involved in the generation of cytokine-responsive Foxp3(-) precursors using studies of mixed bone marrow chimeras as well as TCR-specific generation of Foxp3(+) cells using intrathymic transfer of TCR-transgenic thymocytes expressing a natural Treg TCR. Contrary to a previous report, the analysis of CD28 mutant knockin mice revealed that this cell-intrinsic signal is only partially dependent on the Lck-binding PYAP motif. Surprisingly, even though the absence of CD28 resulted in a 6-fold decrease in thymic Tregs, the TCR repertoires of CD28-deficient and sufficient cells were largely overlapping. Thus, these data suggest that CD28 does not operate by markedly enlarging the repertoire of TCRs available for Treg development, but rather by improving the efficiency of Treg development of thymocytes expressing natural Treg TCRs.

Targeted knock-in mice expressing mutations of CD28 reveal an essential pathway for costimulation.

Despite extensive study, the role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 function has been highly contentious. To definitively address this question, we generated knock-in mice expressing mutations in two critical domains of the cytoplasmic tail of CD28. Mutation of the proximal tyrosine motif interrupted PI3-kinase binding and prevented CD28-dependent phosphorylation of protein kinase B (PKB)/Akt; however, there was no detectable effect on interleukin-2 (IL-2) secretion, expression of Bcl-X(L), or on T-cell function in vivo. Furthermore, we demonstrate that signaling initiated by the C-terminal proline motif is directly responsible for tyrosine phosphorylation of phosphoinosotide-dependent kinase 1, protein kinase C theta, and glycogen synthase kinase 3beta, as well as contributing to threonine phosphorylation of PKB. T cells mutated in this domain were profoundly impaired in IL-2 secretion, and the mice had marked impairment of humoral responses as well as less severe disease manifestations in experimental allergic encephalomyelitis. These data demonstrate that the distal proline motif initiates a critical nonredundant signaling pathway, whereas direct activation of PI3-kinase by the proximal tyrosine motif of CD28 is not required for normal T-cell function.